The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of generation of overt circadian rhythms in mammals. It shows circadian rhythms in glucose utilization and in the frequency of action potential firing, which are maintained even when the SCN is isolated or removed from the animal. The long term aim of this work is to classify SCN neurons according to transmitter content, expression of different sets of ion conductances, synaptic inputs and sensitivity to melatonin, and to measure how these properties change with the time-of-day. Whole-cell patch clamp recordings will be made from SCN neurons in tissue slices removed from rats at different stages of the light-dark cycle. Neurons will be identified by intracellular staining with neurobiotin. the basic ion conductances (calcium, sodium and potassium currents) of the cells will be separated and measured by voltage-dependence, time-dependence and pharmacological blockers. The synaptic inputs to the cells will be evoked by stimulation of the optic nerve, or focal stimulation of the slice: components mediated by excitatory amino acids, gamma-aminobutyric acid, glycine, 5-hydroxytryptamine and neuropeptide Y will be characterized. The action of melatonin and other transmitters on potassium currents and calcium currents will be measured, and the receptors characterized by pharmacological null methods. These properties will be compared among cells at different stages of the light/dark cycle. Later in the project period, methods will be developed for maintaining dissociated SCN cells in culture for several days; circadian rhythms in identified conductances or receptors could be measured on single cells, and blocked by intracellular injection of antibodies of antisense oligonucleotides. The work is expected to contribute to our understanding of the basic mechanisms by which the SCN generates the circadian rhythm, and therefore the ways in which it might be controlled by natural synaptic inputs or by exogenous drugs acting at the membrane of SCN cells. This knowledge will be important for the development of new approaches to the circadian rhythm disorders.